Abstract
Retinoic acid inducible gene-I (RIG-I) ensures immune surveillance of viral RNAs bearing a 5'-triphosphate (5'ppp) moiety. Mutations in RIG-I (C268F and E373A) lead to impaired ATPase activity, thereby driving hyperactive signaling associated with autoimmune diseases. Here we report, using hydrogen/deuterium exchange, mechanistic models for dysregulated RIG-I proofreading that ultimately result in the improper recognition of cellular RNAs bearing 7-methylguanosine and N1-2'-O-methylation (Cap1) on the 5' end. Cap1-RNA compromises its ability to stabilize RIG-I helicase and blunts caspase activation and recruitment domains (CARD) partial opening by threefold. RIG-I H830A mutation restores Cap1-helicase engagement as well as CARDs partial opening event to a level comparable to that of 5'ppp. However, E373A RIG-I locks the receptor in an ATP-bound state, resulting in enhanced Cap1-helicase engagement and a sequential CARDs stimulation. C268F mutation renders a more tethered ring architecture and results in constitutive CARDs signaling in an ATP-independent manner.
Publication types
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Research Support, N.I.H., Extramural
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Research Support, N.I.H., Intramural
MeSH terms
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Adenosine Triphosphatases / metabolism
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Autoimmunity / genetics*
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Caspase Activation and Recruitment Domain / immunology
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DEAD Box Protein 58 / chemistry
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DEAD Box Protein 58 / genetics*
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DEAD Box Protein 58 / immunology
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DEAD Box Protein 58 / metabolism
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Deuterium Exchange Measurement / methods
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Gain of Function Mutation
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Guanosine / analogs & derivatives
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Guanosine / chemistry
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Guanosine / immunology
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Guanosine / metabolism
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Immunity, Innate / genetics*
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Interferon-Induced Helicase, IFIH1 / immunology
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Interferon-Induced Helicase, IFIH1 / metabolism
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Mass Spectrometry / methods
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Methylation
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Models, Molecular
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Mutagenesis, Site-Directed
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Protein Binding / genetics
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Protein Binding / immunology
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RNA Caps / chemistry
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RNA Caps / immunology*
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RNA Caps / metabolism
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RNA, Double-Stranded / chemistry
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RNA, Double-Stranded / immunology*
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RNA, Double-Stranded / metabolism
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RNA, Viral / immunology
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Receptors, Immunologic
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Recombinant Proteins / chemistry
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Recombinant Proteins / genetics
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Recombinant Proteins / immunology
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Recombinant Proteins / metabolism
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Signal Transduction / genetics
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Signal Transduction / immunology
Substances
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RNA Caps
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RNA, Double-Stranded
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RNA, Viral
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Receptors, Immunologic
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Recombinant Proteins
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Guanosine
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7-methylguanosine
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Adenosine Triphosphatases
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RIGI protein, human
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IFIH1 protein, human
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DEAD Box Protein 58
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Interferon-Induced Helicase, IFIH1